Enzyme Lecture 8-22-13
Key Properties of Enzymes
 Enzymes are proteins.
o They can contain non-protein components (cofactors) that participate in
catalysis.
 Enzymes are catalysts.
o Can achieve large enhancements in the rates of reaction.
 Enzymes are highly specific.
o Often stereospecific (distinguish between R and S isomers).
What enzymes do:
 Enzymes do NOT change the equilibrium distribution.
 Enzymes offer alternative reaction pathways with lower activation energies.
 Enzymes accelerate reaction rates in BOTH directions.
 The forward and reverse pathways are the same (“microscopic reversibility”).
Enzyme Classifications—based on dominant feature of the reaction.
 Oxidoreductases – enzymes that catalyze redox reactions.
 Transferases – enzymes that transfer functional groups to a molecule.
 Hydrolases – enzymes that catalyze hydrolysis reactions.
 Lyases – break C-C or C-N through non-hydrolytic/non-oxidative means (generate
double bonds).
 Isomerases – convert between isomers of the same molecule.
 Ligases – covalently join two reactants with the use of energy (usually ATP).
Thermodynamics
Gibbs’ Free Energy
 Independent of reaction path.**
ΔG°’= -RTln([P]/[S]) = Gproduct - Greactant
Reactions of biological interest are at pH 7 and 25°C. Free energy under a given set of
conditions depends on reactant and product concentration, and it reflects the extent of
displacement from equilibrium:
ΔGrxn = ΔG°’ + RTln([P]/[S])
ΔG > 0 Reaction is thermodynamically unfavorable.
ΔG = 0 Reaction is at equilibrium, because RTln([P]/[S]) = -ΔG°’
ΔG < 0 Reaction is thermodynamically favorable, or “spontaneous.”
*In this case, the activation energy must still be overcome.
Coupled reactions
 Energetically unfavorable reactions can be coupled to favorable ones.
o Coupling with the hydrolysis of ATP powers many unfavorable reactions.
Transition State Theory
 All chemical reactions have transition states.
 Transition state = intermediate in structure between reactant and product.
 **Extent to which transition state is populated determines rate of the reaction.
 Energy of activation is the energy difference between reactant and TS.
o Free energy of TS >> free energy of reactant or product.
 Enzymes provide an alternate pathway to reach TS through catalysis.
Enzyme Catalysis
 Enzyme forms a complex with substrate.
 Enzyme is NOT consumed in the reaction.
 Enzyme provides an alternative pathway with lower TS activation energies without
altering standard free energy of the reaction.
 Enzyme increases rate by stabilizing TS.
 **Equilibrium is reached faster but the equilibrium constant (Keq) is not changed.
 Enzymes enhance both forward and reverse reactions
Enzymes and Activation Energy
 ΔG = ΔH – TΔS
o ΔH = change in enthalpy
o T = temperature
o ΔS = change in entropy (ability of system to occupy multiple states)
 ΔH decrease or ΔS increase makes reaction more favorable.
o **Although enzyme lowers entropy by binding to TS, it is partly offset by a
negative ΔH.
Mechanisms of Catalysis
 Proximity
o Reactants are brought together on enzyme surface, increasing local concentration
and orienting reactive groups favorably.
o Enzyme binds TS tighter than substrate.
 *Transition state analogs are good inhibitors
 Strain and Bond Distortion
o Substrate binding can induce a conformational change at active site that induces
strain in substrate.
 **TS may have different structure induced by strain that increases substrate
reactivity.
 Intermolecular (rel rate =1)
 Intramolecular w/ free rotation (rate= 10^5)
 Intramolecular w/ rotation constrained (rate = 10^8)

Acid-Base
o Imidazole (histidyl), carboxyl, amino groups
 Also phenolic (tyrosine), and sulfhydryl (cysteine) residues.

o Protonated forms can act as acid catalysts.
o Deprotonated forms can act as base catalysts.
o Reactivity of AA residues can be affected depending on access to solvent.
 *Hydrophobic regions behave more like organic solvent, whereas hydrophilic
regions may participate more in acid-base catalysis.
Nucleophilic Catalysis
o Nucleophilic catalyst can form a covalent complex with a portion of the substrate.
o In this way, difficult reactions (slow, high Ea reactions) can be divided into
steps with lower Ea’s to speed them up.
o Nucleophilic groups include:
 O of Ser-OH – usually unreactive, but environment can increase reactivity.
 S of Cys – **most reactive protein nucleophile.
 N of Lys or His.
 COO- of Glu and Asp.
o Part of substrate covalently attaches to enzyme and is later released.
 **E-S covalent complex is highly reactive.
 P.250 examples
Active Site -- Area of enzyme that the substrate binds.
 Reactive groups on the enzyme surface are orientated favorably around reactants
 Small part of total enzyme volume.
 Often excludes water to provide a nonpolar (hydrophobic) environment with a lower
dielectric constant.
o Makes electrostatic interactions stronger.
o Substrates can be guided by charge distribution.
o Charge distributions in active sites are thought to stabilize the TS.
 **Noncovalent interactions mediate substrate binding.
o Hydrophobic interactions.
o Hydrogen bonds.
o Ionic interactions.
o Van der Waals forces.
Conformational Flexibility of Enzymes
 Conformation changes when substrate binds.
 Lock and Key Model (Fischer)
o Older concept that explained specificity (but not catalysis).
o Assumes enzymes are rigid.
 Induced Fit (Koshland)
o Explain both catalysis and specificity.
o Enzymes are flexible and change shape to bind to substrate.
Examples of Enzyme Catalysis
 Ribonuclease A (RNAse)
o Hydrolyzes RNA at sites following pyrimidine residues (cytosine/uracil + ribose).
o Cleaves the phosphodiester linkage of the backbone.
o *Requires a 2’OH, so specific for RNA (DNA has no 2’OH).
o Mechanism:
 His12 and His119 act as general acid/base catalysts.
 His12 (acts as base) deprotonates 2’OH, which attacks phosphate.
 Forms a cyclic phosphate intermediate.
 His119 is deprotonated in the process (acts as acid).
 His119 (acts as base) deprotonates H2O, which attacks and breaks the ring.
i. His12 is deprotonated in the process (acts as acid).
**each act as general base and acid

Aspartyl Proteases
o Asp residue functions in acid/base catalysis
o Examples include:
 Pepsin (digestion)
HIV Protease (processes viral polyprotein)
Renin (blood pressure regulation)
 **aspartyl pKa usually 3-4, but pH of blood is ~7
o Mechanism
 Aspartyl residue (general base) abstracts a proton from H2O.
 :OH nucleophilic adds at C=O center of peptide.
 Another aspartyl residue (general acid) protonates the carbonyl oxygen
following nucleophilic addition.
 Aspartyl residue (general base) abstracts a proton from the OH of the former
carbonyl oxygen.
 Electron movement causes peptide bond cleavage.
 The first aspartyl residue (general base) protonates the nitrogen.


Serine Proteases
 Chymotrypsin
o Cleaves at large, hydrophobic residues.
 Trypsin
o Cleaves at arginyl and lysyl residues.
o Has an Asp in the active site that stabilizes basic AA binding.
 Elastase
o Cleaves at small residues (e.g. Ala, Gly).
o Has Val and Thr side chains in the active site that restrict it sterically.
Chymotrypsin Mechanism
 **Specificity determined by cavity that accommodates for side chain of the substrate.
 Seryl, Histidyl and aspartyl residues are essential.
 Ser195 is activated by deprotonation by His57 (acid/base catalysis).
 Nucleophilic attack by Ser195 carbonyl oxygen on peptide bond of substrate.
 Subsequent negative charge of carbonyl oxygen on substrate is stabilized in a cavity called
the “oxyanion hole” by two amide backbone hydrogens.
 His57 serves as general acid and protonates the N of the amide on the substrate.
 Oxyanion reforms carbonyl, kicking out the protonated N.
o **Frees the first part of the peptide.
 His57 deprotonates H2O, allowing nucleophilic attack on acyl-enzyme intermediate.


o Addition-elimination by H2O.
Reforming carbonyl kicks out the Ser.
o **Frees second part of the peptide
Ser-O- deprotonates His57, reforming original catalyst structure.
Prosthetic Groups-- Tightly bound non-protein components needed for enzyme activity.
 Holoenzyme is the active enzyme
o Apoenzyme is the inactive enzyme (protein portion only).
o Holoenzyme = apoenzyme + prosthetic group (cofactor).
 Vitamins are usually precursors to coenzyme substrates used in enzyme reactions.
 Examples:
o Heme
o Flavin



o Thiamine
o Metal ions
Metalloenzymes-- enzymes bound to transition metal ions.
Metal-activated enzymes-- bind metal ions from solution, usually alkali earth metals.
Functions of Metal Ions:
o Complexes with substrates (MgATP)
o Mediates redox reactions through changes in oxidation state
o Stabilizes negative charges with electrostatics
o Promotes OH formation at neutral pH
Effect of Temperature on Enzyme Activity
 Activity often increases about 2-fold per 10°C increase.
 Usually denature at higher temperatures.
o Heat denaturation is normally irreversible due to aggregate formation but many
enzymes will renature after heat induced unfolding
o Enzymes have an optimal temperature range (45-100 C)
Effect of pH on Enzyme Activity
 Activity depends on different groups being ionized and others being protonated during
catalysis.
 Optimal pH range exists, ~7.4 for most enzymes.
Enzyme Kinetics -- rate of enzyme reactions as a function of substrate concentration.
 Michaelis-Menten Kinetics
E + S  ES  E + P
V= (Vmax [S]) / (Km + [S])
Where:
Vmax = maximum velocity of reaction
[S] = concentration of substrate
Km = Michaelis Constant, concentration at which V = ½Vmax
Assumptions:
o Formation of ES is rapid compared to its conversion to E+P.
 We therefore assume that ES is in equilibrium with E+S
o After an initial burst in ES formation, [ES] remains constant.
o [S]>>[E], so S in the form of ES is insignificant when considering [S].
o Reaction rate is proportional to [ES].
o The reverse reaction is negligible.
V= (Vmax [S]) / (Km + [S])
When [S] = Km,
v = 0.5 x Vmax (2nd part of graph)
When [S] >> Km, v = Vmax (3rd part of graph)
When [S] << Km, v = Vmax x [S] / Km (1st part of graph)
See p. 274 for hyperbolic curve
- velocity depends on [S]
Dignam Lecture #2 8-23-13
Steady-state reservoir analogy
 The level in reservoir does Not change b/c it is filled as rapidly as it is
emptied
Briggs-Haldane steady state approximation
 After initial burst of ES formation, rate of ES formation and ES conversion to
E and P are the same
o [ES] is constant as a function of time
V= (Vmax [S]) / (Km + [S])
o Parameter Km is not an equilibrium constant, but a complex kinetic
term
o Briggs-Haldane is more applicable and can describe some systems
where Michaelis-Menten is unsatisfactory
o Inverse reaction becomes
Double Reciprocal (Lineweaver-Burke) Plot
1/v = (Km/Vmax) x 1/[S] + 1/Vmax
o Linear function becomes Y = mx + b

Y= 1/V

m = Km/Vmax (slope)

x = 1/[S]

b = 1/Vmax (y-int)
Enzyme Inhibitors
 inhibition pattern is always stated w/ respect to given substrate
 inhibition patterns in multi-reactant systems may differ depending on
whether A or B is varied (competitive vs non-competitive)

Competitive inhibitors
o Usually resemble substrate
o **Inhibitor and substrate binding are mutually exclusive (only one
binds)
o Do NOT change Vmax
o Can be OVERCOME by raising substrate
o Increase apparent Km for substrate
o Ki = ([E][I]) / [EI]
o Apparent Km increases, affinity decreases
o See graphs p.278

Noncompetitive inhibitors – inhibitor only binds once ES complex has
formed
o Usually do NOT resemble substrate
o May resemble a cosubstrate in multireactant system
o Cannot be OVERCOME by raising substrate
o **Decrease in Vmax, therefore slope increase
o no change in apparent Km
o See graph p. 280

Rate equations in more complex multi-reactant systems
o When product P is present
o Enzyme has 2 substrates (A and B)
 Km terms for A and B
 several possible mechanisms
o Sequential – ordered (one of substrates must bind first) or random
addition of reactants
o Ping pong – product release between addition of reactants; often a
result of covalent intermediate on rxn pathway

Irreversible inhibitors – enzyme is treated w/ reactive reagent that forms a
covalent bond w/ enzyme
o modified enzyme is INACTIVE
o results from modifying an AA involved in catalysis or substrate
binding
o in affinity labels, reagent resembles substrate and covalently
links
 examples: cyanide, diisopropylfluorophosphate
Suicide inhibitors
o Specialized substrates which are converted to irreversible inhibitors
by catalytic action of enzyme

Enzyme regulation
 Substrate levels – availability can limit flux (activity) through a pathway
 Allosteric effectors – may activate or inhibit
 Covalent modification – may activate or inhibit (phosphorylate, adenylate,
methylate)
 Changes in enzyme concentration – may result from changes in rate of
enzyme synthesis (transcription and translation of mRNA)
Allosterism and Cooperativity in Enzymes
 Similar to cooperativity in Hemoglobin
o R and T sites and multiple binding site


o Binding site interactions
Allosteric effectors for enzyme
o may affect Vmax (V type)
o may affect Km (K type)
o Homotropic – substrate binding to a catalytic site causes
conformational changes spread to neighboring subunits (O2 to Hb)
o Heterotropic – ligand binding to a site distinct from the catalytic site
o Feedback inhibition
 end products often inhibit the 1st committed enzyme step in
pathway through binding to allosteric site
Isoenzymes (Isozymes)
o 2 of more different enzymes catalyze same reaction
o can have different kinetic properties
Enzymes and Medicine
 involved in genetic diseases
 drug targets
 diagnostic tools
o How does enzyme defect lead to disease?
 Enzyme activity may be absent b/c of mutation
 inactive, unstable
 defect in mRNA
o reduced mRNA stability or translation
o inactivation of promoter for transcription
 Enzyme activity is deficient – reduced enzyme concentration
or reduced activity
See p. 285 for renin angiotensin system
 Enzymes as therapeutic drugs
o Methotrexate – cancer chemotherapy
o Statin drugs – treat cholesterol
o Beta-lactam antibiotics – bacterial infections
o Ace inhibitors – Angiotensin converting enzyme (ACE) – hypertension
Creatine phosphokinase (CPK) isozymes
 seen in tandem w/ myocardial infarction
Troponins used as Markers for acute MI and heart disease
 troponins – parts of contractile apparatus of muscle that regulate contraction
 see chart p. 290 for myocardial infarction
Myoglobin and Hemoglobin

Key concepts
o Hb is a multifunctional protein that carries O2 and CO2
o Cooperative behavior or proteins facilitates their function
o Point mutations in Hb have effects on function that result in disease
 Important features of Red Blood Cells (RBCs)
o Lack nuclei and mitochondria and do NOT engage in macromolecular
synthesis
o Energy limited to glycolysis and pentose pathway
o Extracellular domains of membrane proteins are sialated, giving
surface significant NEGATIVE charge
 charge prevents aggregation of RBCs and adherence to blood
vessel walls
o Low MW ions and metabolites are exchanged through membrane
transporters
o Cytoskeleton on inner membrane keeps donut shape of RBC
 Hemoglobin in RBCs
o Ionic environment can be controlled independently of conc. in plasma
o Redox environment can be maintained in a reduced state (Fe2+)
Functions of Myoglobin and Hemoglobin in O2 and CO2 Transport and Retention
 Solubility of O2 in water is 75 uM at 37 C, 760 mm Hg, 150 mM NaCl
 Mb increases O2 available in muscle
o [Mb] in red muscle = 500 uM
o capacity for O2 = 500 uM
 Hemoglobin carries O2 from capillaries in lungs to capillaries in tissues
o [Hb] in RBC = 4000 uM
o ***capacity for O2 = 16,000 uM (4 sites on tetramer)


Myoglobin
o *Monomer
o Single heme bound in hydrophobic pocket
o *Single O2 site
o Dominated by alpha helix in a characteristic globin fold
o Histidyl residue that is ligated to iron
Hemoglobin
o *Tetramer (a2B2), 4 heme groups
o *4 O2 sites
o effector sites
 protons
 2,3 BPG
 CO2
 O2
 Clo Structure
 Alpha and Beta form strong aB dimer through hydrophobic
interaction and association is strong
 aB dimers form a2B2 tetramer
 contacts are ionic and polar


association is weak, but tetramer favored in RBCs w/
high concentration of Hb
Globin Fold (alpha type proteins)
o Sequence conservation between different globin fold family members
ranges from 99% to 16%
o 8 a-helices connected by short loops arranged in a pocket structure
for the heme
o hydrophobic nature of buried residues is conserved in different globin
family members
o volume of residues is NOT conserved
 examples: hemoglobin, myoglobin, neuroglobin, phycocyanins
 **not all globin fold type proteins bind heme
O2 Saturation Plot for Myoglobin
 A binding isotherm describes binding of a ligand to a receptor, as the name
implies, at a constant temperature
 See p.341 graph – same hyperbolic curve as Michaelis-Menten
Ligand binding to proteins – single site
 Protein-Ligand <---> Protein + Ligand
 Dissociation constant, Kd = [P] x [L] / [PL]
 [PL] = [Pt] x [L]/ (Kd + [L])
o When [L] << Kd  [PL] = [Pt] x [L] / Kd (initial part of graph)
o When [L] >> Kd  [PL] = [Pt] (last part of graph)
o When [L] = Kd  [PL] = 0.5 [Pt] (middle part of graph)


ligand and acceptor form a complex in equilibrium w/ free acceptor and
ligand
concentration of the complex depends on ligand concentration and Kd
Fractional Saturation (Y)
 concentration of the complex
 at 100% saturation, Y = 1
 Y = [L] / Kd + [L] (general form)
 Y = [MbO2] / [Mbt] = [O2] / (Kd + [O2])
o Kd = 1 Torr = 1 mm Hg
 Effect of [O2] on Y
o When O2 = 0, Y=0
o When O2 = Kd, Y=0.5
o When O2 >> Kd, Y approaches 1
[O2]
Y
0
0
0.5
0.333
1.0
0.5
2.0
.667
(Myoglobin form)
5.0
.833
8.0
.899
10.0
.909
Determination of Kd
 Determine [PL] at several concentrations of [L]
 Several graphic methods
o Hyperbolic saturation plot
o Linear transformations of binding equation (Lineweaver-Burke)
 Linear or nonlinear least squares fit of function to data to get Kd estimates
 Double reciprocal Equation
o 1/[PL] = Kd/[Pt] x 1/[L] + 1/[Pt]
o Y = mx + b
 Y = 1/[PL]
 m = Kd/Pt (slope)
 x = 1/[L]
 b = 1/[Pt]
Hill Approximation for Ligand Binding to Multiple Sites
 Properties
o Equation assumes only unliganded and fully liganded protein
o n = Hill coefficient
o Determination of n gives measure of cooperativity
 n = 1, no cooperativity
 n > 1, positive cooperativity
 n < 1, negative cooperativity
o What does Hill coefficient tell us?
 Has little to do w/ number of ligand binding sites
 Index of extent to which binding is cooperative and whether
cooperativity is (+) or (-)
 Y = [Ln] / Kn + [Ln]
 See graph p.348
o Hill equation for O2 binding to Hb
 Y = [O2]n / Kdn + [O2]n

O2 binding to Mb and Hb
 Mb curve is hyperbolic
o Binds O2 tightly even at very low pressure until
saturation
 50% saturation occurs at 1 torr
 Hb curve is sigmoidal or S-shaped
o Shows cooperative binding – each subsequent
O2 is easier to put on than previous one
 must be able to bind O2 tightly at high
pressure (100 torr)  condition of lungs
 must be able to release O2 at low
pressure (40 torr)  tissues
 Hb gives up O2 where it is needed

Monod (Concerted) Model for Positive Cooperativity
o 2 conformational states:
 R – relaxed, high affinity state
 Oxygenated form
 Left shift
 T – Taut or tense, low affinity state
 Deoxynated form
 Right shift
 Conformers are symmetric (T4 or R4)
 Although affinity of R for ligand is higher than T, w/in T4 or R4
states, affinity is the same for all extents of ligation
 Equilibrium exists between the 2 states